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Microphthalmia and cataract in rats with a novel point mutation in connexin 50 - L7Q.

ABSTRACT: PURPOSE: We isolated an autosomal semi-dominant cataract from our inbred SHR/OlaIpcv rat colony. Heterozygotes express pulverulent cataract with smaller eyes; homozygotes express marked microphthalmia with hypoplastic lens. We call this mutation Dca (for dominant cataract). In this study, we focus on the identification of the responsible gene. METHODS: We performed linkage mapping using 93 F2(SHR-Dca x PD) hybrids and a panel of microsatellite markers. In a separate group of animals with a SHR genetic background, we examined the lenses histologically using Epon semi-thin sections and toluidine blue staining. We also assessed the weight of the eyes as an immediate measure for microphthalmia. RESULTS: We mapped the Dca gene to chromosome 2, spanning 8.6 Mbp between markers D2Rat134 and D2Rat186. By sequencing the most plausible candidate gene, Gja8 (coding for connexin 50), we found a T to A transversion at codon 7, leading to a substitution of glutamine for leucin (L7Q). L7Q lies within the NH(2)-terminal cytosolic domain, presumably involved in voltage gating. Histology revealed disturbances in cell to cell contacts in the lens. CONCLUSIONS: L7Q is a novel mutation in connexin 50 (Gja8), causing semi-dominant pulverulent cataracts. Dca rats can serve as a model for cataract development. A study on the properties of the mutant protein may offer an insight into the connexin channel function.

SUBMITTER: Liska F

PROVIDER: S-EPMC2373795 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Microphthalmia and cataract in rats with a novel point mutation in connexin 50 - L7Q.

Liska Frantisek F Chylíková Blanka B Martínek Jindrich J Kren Vladimír V

Molecular vision 20080507

<h4>Purpose</h4>We isolated an autosomal semi-dominant cataract from our inbred SHR/OlaIpcv rat colony. Heterozygotes express pulverulent cataract with smaller eyes; homozygotes express marked microphthalmia with hypoplastic lens. We call this mutation Dca (for dominant cataract). In this study, we focus on the identification of the responsible gene.<h4>Methods</h4>We performed linkage mapping using 93 F2(SHR-Dca x PD) hybrids and a panel of microsatellite markers. In a separate group of animals ...[more]

PMID: 18470322

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Project description:BACKGROUND:Congenital cataract is the leading cause of blindness in children worldwide. Approximately half of all congenital cataracts have a genetic basis. Protein aggregation is the single most important factor in cataract formation. METHODS:A four-generation Chinese family diagnosed with autosomal dominant congenital cataracts and microphthalmia was recruited at the Shengjing Hospital of China Medical University. Genomic DNA was extracted from the peripheral blood of the participants. All coding exons and flanking regions of seven candidate genes (CRYAA, CRYBA4, CRYBB2, CRYGC, GJA8, MAF, and PITX3) were amplified and sequenced. Restriction fragment length polymorphism (RFLP) assays were performed to confirm the candidate causative variant, c.35G?>?T in the CRYAA gene. We constructed pcDNA3.1(+)-CRYAA expression plasmids containing either the wild-type or the R12L mutant alleles and respectively transfected them into HEK293T cells and into HeLa cells. Western blotting was performed to determine protein expression levels and protein solubility. Immunofluorescence was performed to determine protein sub-cellular localization. RESULTS:A heterozygous variant c.35G?>?T was identified in exon 1 of CRYAA, which resulted in a substitution of arginine to leucine at codon 12 (p.R12L). The nucleotide substitution c.35G?>?T was co-segregated with the disease phenotype in the family. The mutant R12L-CRYAA in HEK293T cells showed a significant increase in the expression level of the CRYAA protein compared with the wild-type cells. Moreover, a large amount of the mutant protein aggregated in the precipitate where the wild-type protein was not detected. Immunofluorescence studies showed that the overexpressed mutant CRYAA in HeLa cells formed large cytoplasmic aggregates and aggresomes. CONCLUSIONS:In summary, we described a case of human congenital cataract and microphthalmia caused by a novel mutation in the CRYAA gene, which substituted an arginine at position 12 in the N-terminal region of ?A-crystallin. The molecular mechanisms that underlie the pathogenesis of human congenital cataract may be characterized by the prominent effects of the p.R12L mutation on ?A-crystallin aggregation and solubility. Our study also expands the spectrum of known CRYAA mutations.

Project description:Several members of connexin family of transmembrane proteins were previously implicated in distinct metabolic conditions. In this study we aimed to determine the effects of complete and heterozygous form of connexin50 gene (Gja8) mutation L7Q on metabolic profile and oxidative stress parameters in spontaneously hypertensive inbred rat strain (SHR).Adult, standard chow-fed male rats of SHR, heterozygous SHR-Dca+/- and SHR-Dca-/- coisogenic strains were used. At the age of 4 months, dexamethasone (2.6 ?g/ml) was administered in the drinking water for three days. The lipidemic profile (cholesterol and triacylglycerol concentration in 20 lipoprotein fractions, chylomicron, VLDL, LDL and HDL particle sizes) together with 33 cytokines and hormones in serum and several oxidative stress parameters in plasma, liver, kidney and heart were assessed.SHR and SHR-Dca-/- rats had similar concentrations of triacylglycerols and cholesterol in all major lipoprotein fractions. The heterozygotes reached significantly highest levels of total (SHR-Dca+/-: 51.3 ± 7.2 vs. SHR: 34.5 ± 2.4 and SHR-Dca-/-: 34.4 ± 2.5 mg/dl, p = 0.026), chylomicron and VLDL triacylglycerols. The heterozygotes showed significantly lowest values of HDL cholesterol (40.9 ± 2.3 mg/dl) compared both to SHR (51.8 ± 2.2 mg/dl) and SHR-Dca-/- (48.6 ± 2.7 mg/dl). Total and LDL cholesterol in SHR-Dca+/- was lower compared to SHR. Glucose tolerance was improved and insulin concentrations were lowest in SHR-Dca-/- (1.11 ± 0.20 pg/ml) in comparison with both SHR (2.32 ± 0.49 pg/ml) and SHR-Dca+/- (3.04 ± 0.21 pg/ml). The heterozygous rats showed profile suggestive of increased oxidative stress as well as highest serum concentrations of several pro-inflammatory cytokines including interleukins 6, 12, 17, 18 and tumor necrosis factor alpha.Our results demonstrate that connexin50 mutation in heterozygous state affects significantly the lipid profile and the oxidative stress parameters in the spontaneously hypertensive rat strain.

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Microphthalmia and cataract in rats with a novel point mutation in connexin 50 - L7Q.

Publications

Microphthalmia and cataract in rats with a novel point mutation in connexin 50 - L7Q.

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